The CA125 antigen is a well known marker for monitoring the recurrence and progression of epithelial ovarian cancer. It is now clear that this antigen is a repeating peptide epitope that is present within the protein backbone of the recently identified mucin MUC16. This mucin is expressed on the surface of ovarian tumor cells (hereafter referred to as csMUC16). A specific proteolytic event results in shedding of MUC16 (hereafter referred to as sMUC16). The sMUC16 can be detected in the serum and is also present in copious amounts in the peritoneal fluid of ovarian cancer patients. The csMUC16 binds to mesothelin, a glycoprotein present on the mesothelial cells and this interaction facilitates peritoneal metastasis of ovarian tumor cells. The sMUC16 present in the peritoneal fluid is unable to inhibit the interaction between csMUC16 and mesothelin. These and other observations suggest specific differences in the biochemical composition of sMUC16 and csMUC16. Because of these structural differences, antibodies that show a preference for csMUC16 isoforms over sMUC16 may be utilized as immunotherapeutic agents against ovarian tumors. Such reagents would have the advantage of targeting the ovarian tumor cells in the presence of high amounts of sMUC16 that is shed in the peritoneal environment. Current immunotherapeutic approaches targeting MUC16 are competed by sMUC16 and thus are limited in their efficacy. The first specific aim of this Phase I STTR proposal is to develop antibodies that preferentially recognize csMUC16. Such specific antibodies will be generated using multiple tolerization immunization approaches. The ABL- MYC technology developed by Neoclone will help us generate an extensive panel of csMUC16 specific antibodies using several different tolerization approaches in a relatively short time period. In the second specific aim, these antibodies will be further characterized for specificity and the relative affinities of the generated antibodies against csMUC16 and sMUC16 will be calculated using Surface Plasmon Resonance and flow cytometry based protocols. The antibodies will be functionally screened for their ability to interrupt the mesothelin interaction. These anti-csMUC16 specific antibodies can then be conjugated with toxins, radiolabels and other agents, or humanized and used as therapeutic agents against ovarian tumors. The therapeutic utility of the antibodies generated will be investigated in the Phase II of this STTR proposal. PUBLIC HEALTH RELEVANCE: Because of the late stage of diagnosis and high rate of recurrence, ovarian cancer is a particularly deadly disease. We propose to develop monoclonal antibodies that will identify unique epitopes specific for a mucin (MUC16) that is highly expressed on ovarian tumors. These antibodies will be extremely useful materials for creation of therapeutic agents that can efficiently target and cytolyze ovarian tumors in the peritoneum.